Influenza A virus (IAV) is an important pathogen. It is responsible for 200,000 hospitalizations and 41,000 deaths in the United States annually. Our previous studies identified that interferon-inducible transmembrane (IFITM) proteins are critical factors for the immune control of IAV in vitro and in vivo. We demonstrated that, uniquely among known viral restriction factors, IFITM proteins restricted viruses before they entered cells. Our overall goal of this proposed project is to understand comprehensively the functions of IFITM proteins and the mechanisms by which these proteins restrict IAV infection. These studies consist of three specific aims. Aim 1. We will determine whether the stability of IFITM3 polymorphisms contribute to their differential control of IAV. We have demonstrated that IFITM3 expression is closely regulated by secretory carrier membrane protein 3 (SCAMP3). We have also noticed that the stability of a recently identified polymorphic IFITM3 ( 21 IFITM3) may explain its correlation to the poor clinical prognosis. In this aim, a series of immunoprecipitatio, immunoblotting, drug inhibitory, and viral entry assays will be performed to determine the pathway contributing to IFITM3 degradation and the mechanism by which SCAMP3 stabilizes its expression. We will also investigate the role of SCAMP3 in the differential control of IAV by wild-type IFITM3 and 21 IFITM3. Aim 2. We will investigate whether IFITM proteins alter intracellular cholesterol homeostasis, resulting in viral entry restriction. In our preliminary studies, we observed that IFITM3 interferes with the homeostasis of intracellular cholesterol via its interaction with vesicle-associated membrane protein-associated protein A (VAPA). In this aim we will clarify whether IFITM-mediated cholesterol accumulation contributes to viral entry restriction. To do so, we will comprehensively characterize the properties of wild-type and functional negative IFITM3 variants and their effects on viral entry under a normal or a cholesterol-depleted condition. We will also evaluate whether this cholesterol accumulation can be similarly induced by IFN. Finally, the effects of cholesterol accumulation on fusion between virion and cell membranes will be determined by confocal imaging. Aim 3. We will examine whether IFITM proteins interfere with the late stage of endocytosis, thereby blocking the progression of virion trafficking. Because IFITM3 also interacts with several vesicle trafficking-related proteins such as CD63, alteration of endosomal trafficking is another potential mechanism of IFITM-mediated restriction. In the aim, we will perform live-cell imaging and cell-free organelle fusion assays to examine whether IFITM proteins interfere with the late stage of endocytosis. The effects of IFITM3 on the composition of endosomal phosphatidylinositol and on the interactions between CD63 and several cellular factors critical for endosomal trafficking (e.g., phosphatidylinositol 4-kinases (PI4K) and adaptor protein 3 (AP-3)) will also be characterized by confocal microscopy and biochemical analyses. We will further evaluate whether PI4K, AP-3, and CD63 alter IFITM- mediated viral entry restriction.